Conventional methods for transforming monocotyledons include the electroporation method, the polyethylene glycol method (PEG method), the particle gun method and so on.
The electroporation method is a method in which protoplasts and the desired DNA are mixed, and holes are formed in the cell membranes by electric pulse so as to introduce the DNA into the cells, thereby transforming the cells. This method currently has the highest reproducibility of the conventional methods and various genes have been introduced into monocotyledons, especially into rice plants by this method (Toriyama K. et al., 1988; Bio/Technol. 6:1072-1074, Shimamoto K. et al., 1989; Nature 338:274-276, Rhodes C. A. et al., 1989; Science 240:204-207). However, this method has the problems that 1) it can be applied only to the plant species for which the system for regenerating plants from protoplasts has been established, 2) since it takes several months to regenerate plants from the protoplasts, a long time is required to obtain transformants, and that 3) since the culture period is long, the frequency of emergence of mutants during the culture is high accordingly, so that the probability of obtaining normal transformants is decreased.
The PEG method is a method in which the desired gene and protoplasts are mixed and the mixture is treated with PEG, thereby introducing the gene into the protoplasts. This method is different from the electroporation method in that PEG is used instead of the electric pulse. The efficiency of introducing the gene is thought to be somewhat lower than the electroporation method. Although there is a report that transformants were obtained by this method, this method is not widely used. Since protoplasts are used, this method has the same problems as in the electroporation method (Zhang W. et al., 1988; Theor. Appl. Genet. 76:835-840, Datta S. K. et al., 1990; Bio/Technol. 8:736-740).
The particle gun method is a method in which the desired gene is attached to fine metal particles, and the metal particles are shot into cells or tissues at a high speed, thereby carrying out the transformation. Thus, according to this principle, transformation may be performed on any tissues. Therefore, this method is effective for transforming the plant species for which the systems for regenerating plants from protoplasts have not been established. The efficiency of transformation varies depending on the selection after the gene was shot. There is no data which compare the efficiency of this method with that of the electroporation method (Gordon-Kamm W. J. et al., 1990; Plant Cell 2:603-618, Fromm M. E. et al., 1990; Bio/Technol. 8:833-839, Christou P. et al., 1991; Bio/Technol. 9:957-962).
Other methods include 1) culturing seeds or embryos with DNA (Topfer R. et al., 1989; Plant Cell 1:133-139, Ledoux L. et al., 1974 Nature 249:17-21); 2) treatment of pollen tube (Luo and Wu 1988; Plant Mol. Biol. Rep. 6:165-), 3) liposome method (Caboche M. 1990; Physiol. Plant. 79:173-176, Gad A. E. et al., 1990:177-183) and 4) microinjection method (Neuhaus G. et al., 1987; Theor. Appl. Genet. 75:30-36). However, these methods have problems in the efficiency of transformation, reproducibility or applicability, so that these methods are not commonly used.
On the other hand, a method for introducing a gene using the Ti plasmid of bacteria belonging to the genus Agrobacterium as a vector is widely used for transforming dicotyledons such as tobacco, petunia, rape and the like. However, it is said that the hosts of the bacteria belonging to genus Agrobacterium are restricted to dicotyledons and that monocotyledons are not parasitized by Agrobacterium (De Cleene M. 1976; Bot. Rev. 42:389-466).
As for transformation of monocotyledons by Agrobacterium, although transformation of asparagus (Bytebier B. et al., 1987: Proc. Natl. Acad. Sci. USA, 84:5345-5349) and of Dioscorea bulbifera (Schafew et al., 1987; Nature 327:529-532) has been reported, it is said that this method cannot be applied to other monocotyledons, especially to the plants belonging to family Gramineae (Potrykus I. 1990; Bio/Technol. 8:535-543).
Grimsley et al. (1987: Nature 325:177-179) reported that T-DNA of Agrobacterium in which DNA of maize streak virus was inserted was inoculated to the apical meristem of maize plants and infection of the plants by maize streak virus was confirmed. Since the infected symptoms are not observed when merely the DNA of maize streak virus is inoculated, they interpreted the above-mentioned result as a piece of evidence showing that Agrobacterium can introduce DNA into maize. However, since it is possible that a virus replicates even if it is not incorporated into the nucleus genome, the result does not show that the T-DNA was incorporated into the nucleus. They subsequently reported that the infection efficiency is the highest when the virus is inoculated to the apical meristem in the shoot apex of the maize (Grimsley et al., 1988: Bio/Technol. 6:185-189), and that virC gene in the plasmid of Agrobacterium is indispensable to the infection (Grimsley et al., Mol. Gen. Genet. 217:309-316).
Gould J. et al. (1991; Plant Physiol. 95:426-434) inoculated super-virulent Agrobacterium EHA1 having a kanamycin-resistant gene and a GUS gene to shoot apices of maize after injuring the shoot apices with a needle, and selected the shoot apex based on the resistance to kanamycin. As a result, plants having resistance to kanamycin were obtained. They confirmed by Southern blotting analysis that some of the seeds of the subsequent generation of the selected plants had the introduced gene (chimera phenomenon).
Mooney P. A. et al., (1991; Plant Cell, Tissue, Organ Culture 25:209-218) tried to introduce the kanamycin-resistance gene into embryos of wheat using Agrobacterium. The embryos were treated with an enzyme to injure the cell walls, and then Agrobacterium was inoculated. Among the treated calli, although a very small number of calli which were assumed to be transformants grew, plants could not be regenerated from these calli. The existence of the kanamycin-resistance gene was checked by Southern blotting analysis. As a result, in all of the resistant calli, change in structure of the introduced gene was observed.
Raineri et al. (1990; Bio/Technol. 8:33-38) inoculated super-virulent Agrobacterium A281 (pTiBo542) to 8 varieties of rice after injuring the scutella of the rice plants. As a result, growth of tumor-like tissues was observed in two varieties, Nipponbare and Fujisaka 5. Further, an Agrobacterium containing a plasmid having a T-DNA from which a hormone-synthesizing gene was removed and instead, a kanamycin-resistant gene and GUS gene were inserted therein was inoculated to embryos of rice. As a result, growth of kanamycin-resistant calli was observed. Although the expression of GUS gene was observed in these resistant calli, transformed plants could not be obtained from the calli. They interpreted these results as that the T-DNA was introduced into rice cells.
Thus, although the experimental results which suggest that introduction of genes into the plants belonging to family Gramineae such as rice, maize and wheat can be attained by using Agrobacterium have been reported, fully convincing results have not been obtained about the reproducibility, introduction efficiency and about the confirmation of the introduction of the gene (Potrykus I. 1990; Bio/Technol. 8:535-543).
As mentioned above, introduction of genes into the plants belonging to the family Gramineae is now mainly carried out by the electroporation method. However, with this method, since protoplasts are used, a long time and much labor are required to obtain regenerated plants. Further, there is a danger that mutants may emerge at a high frequency due to the long culturing period. Still further, this method cannot be applied to the plants such as maize for which the system for regenerating plants from protoplasts has not been established. In view of this, as mentioned above, as for maize, use of the apical meristem has been tried. However, the operation for isolating the apical meristem requires much labor and it is not easy to prepare apical meristems in a large amount.